The cyclic AMP (cAMP) receptor protein (CRP) is a DNA binding protein from Escherichia coli that can activate transcription when bound to specific sites located near promoters. CRP can serve as a model system for the study of gene regulation. We have been studying several steps in the pathway to CRP activation of transcription. (i) In order for CRP to become activated as a DNA binding protein, cAMP must bind to the protein and cause an allosteric change. We have isolated CRP mutants that are defective in the cAMP-induced allosteric change. We are initiating a collaboration with Richard Brennan at the Oregon Health Sciences University, for X-ray crystallographic analysis of one of the CRP mutants. (ii) We have found that the molecular chaperone DnaK aids in achieving CRP stability in vivo. This result may partially explain why CRP-mediated transcription activation is always greater in vivo than in vitro. (iii) We have addressed the mechanism for transcription activation by CRP by three different approaches. A previous model for CRP activation of transcription stated that CRP activated by placing the a subunit of RNA polymerase down on the DNA in a particular manner. By constructing a promoter with both CRP binding sites and an a binding site, we have shown that CRP activation of transcription is independent of a placement on DNA. We have isolated mutations in the crp gene that allow CRP to activate transcription from a longer distance from the promoter, and are currently analyzing them. Also, we have continued our studies on transcription activation on DNA that has a disruption between where CRP binds and the promoter. This disrupted DNA does not allow CRP activation of transcription because unwinding of the DNA at the promoter does not occur. We have also developed a protein footprinting assay that allows identification of regions within a protein involved in interacting with DNA or other proteins.